Multilevel interrogation of H3.3 reveals a primordial role in transcription regulation

Background Eukaryotic cells can rapidly adjust their transcriptional profile in response to molecular needs. Such dynamic regulation is, in part, achieved through epigenetic modifications and selective incorporation of histone variants into chromatin. H3.3 is the ancestral H3 variant with key roles in regulating chromatin states and transcription. Although H3.3 has been well studied in metazoans, information regarding the assembly of H3.3 onto chromatin and its possible role in transcription regulation remain poorly documented outside of Opisthokonts. Results We used the nuclear dimorphic ciliate protozoan, Tetrahymena thermophila, to investigate the dynamics of H3 variant function in evolutionarily divergent eukaryotes. Functional proteomics and immunofluorescence analyses of H3.1 and H3.3 revealed a highly conserved role for Nrp1 and Asf1 histone chaperones in nuclear influx of histones. Cac2, a putative subunit of H3.1 deposition complex CAF1, is not required for growth, whereas the expression of the putative ortholog of the H3.3-specific chaperone Hir1 is essential in Tetrahymena. Our results indicate that Cac2 and Hir1 have distinct localization patterns during different stages of the Tetrahymena life cycle and suggest that Cac2 might be dispensable for chromatin assembly. ChIP-seq experiments in growing Tetrahymena show H3.3 enrichment over the promoters, gene bodies, and transcription termination sites of highly transcribed genes. H3.3 knockout followed by RNA-seq reveals large-scale transcriptional alterations in functionally important genes. Conclusion Our results provide an evolutionary perspective on H3.3’s conserved role in maintaining the transcriptional landscape of cells and on the emergence of specialized chromatin assembly pathways. Supplementary Information The online version contains supplementary material available at 10.1186/s13072-023-00484-9.


Multiple sequence alignment and Phylogenetic analysis
Protein sequences for the HHT2 and HHT3 genes were obtained from the Tetrahymena Genome Database (www.ciliate.org) and aligned using PRALINE alignment toolbox (Simossis and Heringa 2005).
Protein sequences were retrieved using BLAST searches using NCBI non-redundant protein sequences database (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Protein sequences were analyzed at the Pfam (http://pfam.sanger.ac.uk/) and SMART (http://smart.embl-heidelberg.de/) databases to examine the domain architecture (Finn et al. 2016;Letunic and Bork 2018). Protein phylogenetic analysis were carried out using the neighbour-joining method under p-distances using MEGA 7 (Kumar et al. 2016). Multiple sequence alignments were constructed using MUSCLE with default parameters. The reliability of the resulting phylogenetic trees was assessed using the bootstrap method (1000 replicas for each tree).
Incubation was for 15min in a 65 • C water bath with brief vortexing every few minutes. 450ul of elution buffer was also added to the thawed input samples. 20ul of 5M NaCl was added and the samples were incubated overnight at 65 • C for reverse crosslinking. Samples were treated with RNase (Fermentas) and incubated for 1h at 37ᵒC. Proteinase K (Fermentas) was added and incubated at 42ᵒC for 2 hrs and 65ᵒC for 8hrs. The DNA was isolated (Qiagen PCR purification kit) and H2O was used for elution.

Mass Spectrometry sample preparation
Preparation of protein eluates for mass spectrometry acquisition was essentially as previously described (Saettone et al. 2018;Nabeel-Shah, Garg, Kougnassoukou Tchara, et al. 2021) and is provided below as it is. Briefly, the eluates were dried using a speed vacuum apparatus and resuspended in 10μL of 20 mM Tris-HCl pH 8.0. Trypsin digestion was carried out using 0.75 μg of trypsin (Sigma) for ~ 15 hours at 37°C with mild agitation. An extra 0.25 μg of trypsin was added to each sample and incubated for an additional 3 h. The samples were acidified to a final concentration of 2% acetic acid, desalted using C18StageTips (Thermo Scientific) as per the manufacturer's instructions and stored at -80°C until their acquisition on a mass spectrometer.
Mass spectrometry acquisition using Triple TOF 5600 mass spectrometer 5 μL of each sample, representing 50% of the sample, was directly loaded at 300 nL/min onto a New Objective PicoFrit column (15 cm×0.075 mm I.D; Scientific Instrument Services, Ringoes, NJ) packed with Jupiter 5 μm C18 (Phenomenex, Torrance, CA) stationary phase. The peptides were eluted from the column by a gradient generated by an Agilent 1200 HPLC system (Agilent, Santa Clara, CA) equipped with a nano electrospray ion source coupled to a 5600+ Triple TOF mass spectrometer (Sciex, Concord, ON). A 65-min linear gradient of a 5-35% mixture of 0.1% formic acid injected at 300 nL/min was used to elute peptides. Data dependent acquisition mode was used in Analyst version 1.7 (Sciex) to acquire mass spectra. Full scan mass spectrum (400 to 1250m/z) were acquired followed by collision-induced dissociation of the twenty most intense ions. A period of 20 s and a tolerance of 100 ppm were set for dynamic exclusion.

Mass spectrometry acquisition using Orbitrap Fusion mass spectrometer
Peptide samples were separated by online reversed-phase (RP) nanoscale capillary liquid chromatography (nanoLC) and analyzed by electrospray mass spectrometry (ESI MS/MS). The experiments were performed with a Dionex UltiMate 3000 nanoRSLC chromatography system (Thermo Fisher Scientific) connected to an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific) equipped with a nanoelectrospray ion source. Peptides were trapped at 20 ul / min in loading solvent (2% acetonitrile, 0.05% TFA) on a 5mm x 300 μm C18 pepmap cartridge precolumn (Thermo Fisher Scientific) during 5 minutes. The pre-column was switched online with a self-made 50 cm x 75 µm internal diameter separation column packed with ReproSil-Pur C18-AQ 3-μm resin (Dr. Maisch HPLC) and the peptides were eluted with a linear gradient from 5-40% solvent B (A: 0,1% formic acid, B: 80% acetonitrile, 0.1% formic acid) in 60 minutes, at 300 nL/min. Mass spectra were acquired using a data dependent acquisition mode using Thermo XCalibur software version 3.0.63. Full scan mass spectra (350 to 1800m/z) were acquired in the orbitrap using an AGC target of 4e5, a maximum injection time of 50 ms and a resolution of 120 000. Internal calibration using lock mass on the m/z 445.12003 siloxane ion was used. Each MS scan was followed by acquisition of fragmentation spectra of the most intense ions for a total cycle time of 3 seconds (top speed mode). The selected ions were isolated using the quadrupole analyzer in a window of 1.6 m/z and fragmented by Higher energy Collision-induced Dissociation (HCD) with 35% of collision energy. The resulting fragments were detected by the linear ion trap in rapid scan rate with an AGC target of 1E4 and a maximum injection time of 50ms. Dynamic exclusion of previously fragmented peptides was set for a period of 20 sec and a tolerance of 10 ppm.

Mass spectrometry acquisition using LTQ mass spectrometer
The suspended sample was bomb-loaded in its entirety on the equilibrated column. The column was washed off-line for 10 min in buffer A and placed in-line with a LTQ mass spectrometer equipped with an Agilent 1100 pump with split flow, and either the Thermo source, or a Proxeon source. Buffer A is 2% acetonitrile (ACN), 0.1% formic acid; buffer B is 98% ACN, 0.1% formic acid. The HPLC gradient program delivered an ACN gradient over 120 min (1-5% buffer B over 4 min, 5-40% buffer B over 100 min, 40-60% buffer B over 5 min, 60-100% buffer B over 5 min, hold buffer B at 100% 3 min, and 100-0%B in 2 min). The parameters for Data Dependent Acquisition on the mass spectrometer were: 1 centroid MS (mass range 400-2000) followed by MS/MS on the 5 most abundant ions. General parameters were activation type = CID, isolation width = 3, normalized collision energy = 32, activation Q = 0.25, activation time = 30 msec, wide band activation. The minimum threshold was 1000, the repeat4count = 1, repeat duration = 30 sec, exclusion size list = 500, exclusion duration = 30sec, exclusion mass width (by mass) = low 1.2, high 1.5.

Data Dependent Acquisition MS analysis:
Mass spectrometry data were stored, searched and analyzed using the ProHits laboratory information management system (LIMS) platform (Liu et al. 2016). Within ProHits, Thermo Fisher scientific RAW mass spectrometry files were converted to mzML and mzXML using ProteoWizard (3.0.4468; (Kessner et al. 2008). Within ProHits, AB SCIEXWIFF files were first converted to an MGF formatusing WIFF2MGF converter and to an mzML format using ProteoWizard (v3.0.4468) and the AB SCIEX MS Data Converter (V1.3 beta). The mzML and mzXML files were searched using Mascot (v2.3.02). The spectra were searched with the RefSeq database (version45, January 24th, 2011) acquired from NCBI against a total of 24,770 T. thermophila sequences. For TripleTOF files, the database parameters were set to search for tryptic cleavages, allowing up to two missed cleavage sites per peptide with a mass tolerance of 40 ppm for precursors with charges of 2+ to 4+ and a tolerance of +/-0.15 amu for fragment ions. For Orbitrap Fusion files, the database parameters were set to search for tryptic cleavages, allowing up to two missed cleavage sites per peptide with a mass tolerance of 12 ppm for precursors with charges of 2+ to 4+ and a tolerance of +/-0.6 amu for fragment ions. For files analyzed on the LTQ, the charges +1, +2 and+3 were considered, with the parent mass tolerance set at 3 amu and the fragments at 0.6 amu. Deamidated asparagine and glutamine and oxidized methionine were